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The purpose of this communication is to elucidate if selenium plays a role in the function of granulocytes and lymphocytes. Thus, the incorporation of selenium in proteins from granulocytes and lymphocytes cultured with 1 microCi/mL radioactive Na2(75)SeO3 was studied. The protein peaks containing 75Se from two columns of Heparin Sepharose CL-6B and Sephacryl S-200 HR were separated further by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The results showed that the incorporation of 75Se into granulocytes was about six times higher than that of lymphocytes during a 96-h cultivation, however, the GSH-Px activity in granulocytes did not change significantly. On the other hand, the GSH-Px activity of lymphocytes rose significantly after three days cultivation. These data indicated that the main chemical form of selenium in granulocytes was not GSH-Px. Results from SDS-PAGE revealed a strongly 75Se-labeled protein band with subunit molecular weight of 15 kDa in the supernatant of granulocyte homogenate. However, the main chemical forms of selenium in the culture media of granulocytes and lymphocytes were found to be selenoprotein P. The different forms of selenium-containing proteins in the intracellular and extracellular media of granulocytes indicated the different functions of these proteins.
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PMID:Different selenium-containing proteins in the extracellular and intracellular media of leucocytes cultivated in vitro. 953 63

We have established and characterised a cell line, designated WART, from a patient with primary adenocarcinoma of the lung. This cell line grows with a doubling time of approximately 15 hours, forms colonies in soft agarose, is tumorigenic in athymic nude mice, and has a complex karyotype with both structural and numerical abnormalities. WART serum free conditioned medium (SFCM) contains a factor which stimulates motile behavior of WART cells. This factor with an apparent molecular weight of 67 kDa induced in an autocrine fashion prominent pseudopodia, and chemotactic and chemokinetic responses. Heparin affinity chromatography, ion exchange and molecular sieve chromatography accompanied by SDS-PAGE analysis showed that the motility inducing activity was associated with a major band with molecular weight 67 kDa. The motility inducing activity of the 67 kDa protein was not sensitive to reduction with either dithiotreitol or mercaptoethanol which distinguishes it from A-2058 melanoma autocrine motility factor (AMF)/autotaxin, HT-1080 fibrosarcoma AMF and scatter factor which lose their biological activity upon reduction. This 67 kDa motility inducing factor did not augment DNA synthesis indicating that its locomotor activity is independent of mechanisms regulating cell growth. Pertusis toxin inhibited the motile response induced by the 67 kDa protein indicating a signal transduction pathway involving G proteins. Due to its production of the motility stimulating protein the cell line could facilitate studies of invasion and metastasis of human lung tumors.
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PMID:Production of a motility factor by a newly established lung adenocarcinoma cell line. 961 17

Quantitative characterization of the interaction of des-kringle1-5-plasmin (microplasmin) with fibrin(ogen) and plasma protease inhibitors may serve as a tool for further evaluation of the role of kringle domains in the regulation of fibrinolysis. Comparison of fibrin(ogen) degradation products yielded by plasmin, miniplasmin (des-kringle1-4-plasmin), microplasmin, and trypsin on SDS gel electrophoresis indicates that the differences in the enzyme structure result in different rates of product formation, whereas the products of the four proteases are very similar in molecular weight. Kinetic parameters show that plasmin is the most efficient enzyme in fibrinogen degradation, and the kcat/KM ratio decreases in parallel with the loss of the kringle domains. The catalytic sites of the four proteases have similar affinities for fibrin (KM values between 0.12 and 0.21 microM). Trypsin has the highest catalytic constant for fibrin digestion (kcat = 0.47 s-1), and among plasmins with different kringle structures, the loss of kringle5 results in a markedly lower catalytic rate constant (kcat = 0.0076 s-1 for microplasmin vs 0.048 s-1 for miniplasmin and 0.064 s-1 for plasmin). In addition, microplasmin is inactivated by plasmin inhibitor (k" = 3.9 x 10(5) M-1 s-1) and antithrombin (k" = 1.4 x 10(3) M-1 s-1) and the rate of inactivation decreases in the presence of fibrin(ogen). Heparin (250 nM) accelerates the inactivation of microplasmin by antithrombin (k" = 10.5 x 10(3) M-1 s-1 ), whereas that by plasmin inhibitor is not affected (k" = 4.2 x 10(5) M-1 s-1).
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PMID:Fibrinolysis with des-kringle derivatives of plasmin and its modulation by plasma protease inhibitors. 963 57

Human plasma-derived von Willebrand factor (hp-vWF) and recombinant von Willebrand factor (r-vWF) have been fractionated by heparin affinity chromatography followed by multimer analysis using SDS-agarose gel electrophoresis. Because heparin binding sites are contained in each vWF subunit, high molecular weight multimers of r-vWF and hp-vWF, respectively, were eluted with higher salt concentration, in comparison to r-vWF and hp-vWF molecules with a low degree of multimerization. Heparin affinity chromatography did not affect the multimer composition of r-vWF. By contrast, faster migrating satellite bands and slower migrating satellite bands of hp-vWF exhibited reduced and increased heparin affinity, respectively, compared to the intermediate band of the same triplet. Because heparin binding sites are localised in the N-terminal domain of the hp-vWF subunit, this result confirms a structural model of hp-vWF (Fischer et al., Biochem. J. 1998;331:483-488) suggested recently, in which the slower migrating satellite bands have excess of one N-terminal fragment and the faster migrating satellite bands lack one N-terminal fragment, respectively, in comparison with the corresponding intermediate triplet band.
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PMID:Selectivity of von Willebrand factor triplet bands towards heparin binding supports structural model. 1008 94

A new hetero-bifunctional photo crosslinking reagent, 2-(4-azidoanilyl)-4-(4-azabicyclo-[2,2, 2]hexylammonio)-6-morpholino-1,3,5-triazine chloride, was designed to detect and isolate heparin-binding protein(s) that may act as heparin-receptor(s) on the platelet surface. In a preliminary study using ethanol as a model substrate, the reagent was shown to react with the alcoholic hydroxy group under mild conditions and its crosslinking photoreactivity was high. The reagent effectively formed similar covalent bonds with heparin, while preserving its anticoagulant anti-Xa activity. [3H]Heparin labeled with this reagent crosslinked to antithrombin III very specifically but not to ovalbumin, as analyzed by the Bio-imaging Analyzer System (BAS, Fuji Photo Film, Tokyo). Affinity crosslinking of [3H]heparin was then used to detect heparin-binding proteins on the surface of intact platelets. Several discrete protein bands were detected by the BAS-imaging of SDS-PAGE.
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PMID:A novel crosslinking reagent and its application for the detection and isolation of heparin-binding protein(s) on the platelet surface. 1034

Aggregation and precipitation are major pitfalls during bioprocessing and purification of recombinant human basic fibroblast growth factor (rh-bFGF). In order to gain high yields of the soluble protein monomer with high biological activity, an efficient downstream process was developed, focussing on the combination of expanded bed adsorption (EBA) and heparin chromatography. After expression in E. coli TG1:plambdaFGFB, cells were harvested and washed; then the rh-bFGF was released via high pressure homogenization. The high viscosity of the feedstock of about 40 mPa s, showing non-newtonian behaviour, was reduced to 2 mPa s by the addition of DNase. The homogenate (5.6 l) was loaded directly on an expanded bed column (C-50) packed with the strong cation-exchanger Streamline SP. In the eluates, histone-like (HU) protein was identified as the main protein contaminant by sequence analysis. The thermodynamics and kinetics of rh-bFGF adsorption from the whole broth protein mixture were determined in view of competition and displacement effects with host-derived proteins. Optimal binding and elution conditions were developed with knowledge of the dependence of rh-bFGF adsorption isotherms on the salt concentration to allow direct application of eluates onto Heparin HyperD. This affinity support maintained selectivity and efficiency under CIP and over a wide range of flow-rates; both is advantageous for the flexibility of the purification protocol in view of a scalable process. Remaining DNA and HU protein were separated by Heparin HyperD. The endotoxin level decreased from approximately 1,000,000 EU/ml in the whole broth to 10 EU in 3 mg bFGF per ml. The final purification protocol yields >99% pure rh-bFGF as judged from SDS-PAGE and MALDI-TOF mass spectrometry with high mitogenic activity (ED50=1-1.5 ng/ml) of the lyophilized sample. In comparison to the conventional process, the overall protein recovery rose by 15% to 65% with saving time and costs.
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PMID:Preparative two-step purification of recombinant human basic fibroblast growth factor from high-cell-density cultivation of Escherichia coli. 1068 Oct 38

A novel trypsin-type serine proteinase, which processes the precursors of the envelope fusion glycoproteins of pneumotropic Sendai and human influenza A viruses, was purified to homogeneity from pig lungs. On SDS/PAGE, the purified enzyme gave a protein band corresponding to about 32 kDa, and has an apparent molecular mass of 120 kDa, as determined by gel permeation chromatography. Immunohistochemical staining with antibodies against this enzyme revealed that the enzyme is located in pig lung mast cells. The N-terminal 44-amino-acid sequence of the enzyme exhibits about 80% identity with those of mast cell tryptases from other species. Of the inhibitors tested, di-isopropyl fluorophosphate, antipain, leupeptin, benzamidine and a few proteinaceous inhibitors, such as mucus protease inhibitor and aprotinin, inhibited this enzyme activity. Heparin stabilized the enzyme, but high-ionic-strength conditions did not, unlike for human mast cell tryptase. The purified enzyme efficiently processed the fusion glycoprotein precursor of Sendai virus and slowly processed hemagglutinin of human influenza A virus, and triggered the infectivity of Sendai virus in a dose-dependent manner, although human mast cell tryptase beta and rat mast cell tryptase (rat MCP-7) from lungs did not process these fusion glycoproteins at all. These results suggest that mast cell tryptase in pig lungs is the possible trigger of the pneumotropic virus infections.
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PMID:Mast cell tryptase from pig lungs triggers infection by pneumotropic Sendai and influenza A viruses. Purification and characterization. 1082 3

Midkine is a heparin-binding growth factor with survival-promoting and migration-enhancing activities. In order to understand the regulation of midkine signaling, we isolated midkine-binding proteoglycans from day 13 mouse embryos, when midkine is intensely expressed. Deglycosylation followed by SDS/PAGE revealed various protein bands; one of these was identified as PG-M/versican by in gel trypsin digestion and sequencing the resulting peptides. PG-M/versican isolated from day 13 mouse embryos bound midkine with a Kd of 1.0 nM. Pleiotrophin/heparin-binding growth-associated molecule, which has a structure related to midkine, was also bound similarly. Digestion with chondroitinase ABC, AC-I or B abolished the binding to midkine. Heparin as well as chondroitin sulfate D and E inhibited the binding. After chondroitinase ABC digestion, the midkine-binding PG-M/versican released 4-sulfated, 6-sulfated, 2, 6-disulfated and 4,6-disulfated unsaturated disaccharides. These results suggest that midkine binds to a polysulfated domain in the chondroitin sulfate chain with a region of dermatan sulfate structure. This proteoglycan may modulate the midkine activity, as binding to midkine can enhance midkine action by concentrating it to the cell periphery or inhibit the action by competing with the binding to a signaling receptor.
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PMID:A heparin-binding growth factor, midkine, binds to a chondroitin sulfate proteoglycan, PG-M/versican. 1086 5

The porcine reproductive and respiratory syndrome virus (PRRSV) has a very restricted tropism for well-differentiated cells of the monocyte-macrophage lineage, which is probably determined by specific receptors on these cells. In this study, the importance of heparinlike molecules on porcine alveolar macrophages (PAM) for PRRSV infection was determined. Heparin interacted with the virus and reduced infection of PAM up to 92 or 88% for the American and European types of PRRSV, respectively. Other glycosaminoglycans, similar to heparin, had no significant effect on infection while heparinase treatment of PAM resulted in a significant reduction of the infection. Analysis of infection kinetics showed that PRRSV attachment to heparan sulfate occurs early in infection. A heparin-sensitive binding step was observed which converted completely into a heparin-resistant binding after 120 min at 4 degrees C. Using heparin-affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), it was observed that the structural matrix (M) and nucleocapsid (N) proteins attached to heparin. Nonreducing SDS-PAGE revealed that M bound to heparin mainly as a complex with glycoprotein GP(5) and that the N protein bound to heparin as a homodimer. GP(3), which was identified as a minor structural protein of European types of PRRSV, did not bind to heparin. Since the N protein is not exposed on the virion surface, it was concluded that the structural M protein and the M-GP(5) complex contribute to PRRSV attachment on a heparinlike receptor on PAM. This is the first report that identifies a PRRSV ligand for a cell surface heparinlike receptor on PAM.
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PMID:Involvement of the matrix protein in attachment of porcine reproductive and respiratory syndrome virus to a heparinlike receptor on porcine alveolar macrophages. 1193 97

The human VEGF(165) cDNA was amplified by PCR, and was inserted, after confirming by DNA sequence analysis, into the Pichia pastoris expression vector pPIC9K containing AOX1 promoter and lead sequence of alpha factor gene to form a recombinant expression plasmids pPIC9K/hVEGF(165). This recombinant plasmid was transformed into KM71. Transformants were screened, cultured inflasks and induced by the addition of 1% methanol. After 4 days of methanol induction, the expressed hVEGF(165) came up to 60% of total proteins in medium supernatant as shown by SDS-PAGE. Western blot assay proved that the expressed hVEGF(165) had good antigenicity and high specificity. The recombinant protein was further purified by using Heparin-Sepharose CL6B affinity chromatography, and was proved that it had good biological activity to stimulate HUVEC proliferation and to promote collateral blood vessel formation in an acquired limb artery occlusion animal model.
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PMID:Expression, Purification and Biological Activity Analysis of Human Vascular Endothelial Growth Factor (VEGF(165)) in Pichia pastoris. 1205 Jul 94


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